The infestations by nematode parasites of animals such as sheep and cattle are of economic importance to those in the agriculture industry. Traditionally, nematode infection has been treated by the administration of anthelmintics.
However, a major drawback with conventional anthelmintics is that nematode resistance to a broad spectrum of anthelmintics is now becoming increasingly more widespread and is therefore of serious concern (Waller, 1997; Sangster et al, 1999; Van Wyk et al, 1999)
A number of mechanisms have been proposed to explain expulsion of nematodes from the intestine of immune sheep (Rothwell 1989). There is evidence for the involvement of elements of an immediate hypersensitivity response, where antigenic stimulation of IgE-sensitised mucosal mast cells leads to an accumulation of substances in mucus which may affect nematode survival (Miller 1996; Emery et al 1997). Anti-nematode properties of mucus include the presence of chemical mediators (Douch et al 1983; Jones et al 1990) and antibody (Lee & Ogilvie 1981; Miller 1987; Carlisle et al 1991). Recently, the present inventors showed that intestinal mucus obtained from sheep immunised by multiple truncated infections could alter the normal pattern of larval establishment after infusion of a mixture of larvae and mucus into the duodenum of naive recipient sheep (Harrison et al 1999). Mucus collected from the small intestine of sheep immune to the parasitic nematode Trichostrongylus colubriformis was found to have anti-larval activity, causing larvae to clump in vitro and resulting in significant reduction of numbers of larvae establishing in naive sheep after infusion of larvae and mucus via a duodenal cannula.
Immunoblotting showed that immune mucus contained IgG and IgA antibodies that recognised predominantly an antigen with an estimated molecular weight of 35 kDa. Antibodies eluted from the surface of larvae incubated in immune mucus also reacted with the 35 kDa antigen on blots of larval homogenate. Immunofluorescence and immunogold electron microscopy showed that the 35 kDa antigen was present on the epicuticle of L3 and was shed during the moult to L4. The antigen was not present in eggs, L1, L2, L4 or adult worms and was only seen in extracts of L3 before infection and up to 5 days after infection. The results suggest that the binding of antibody to the larval surface prevented larvae from establishing at their preferred site, causing them to be eliminated from the intestine. Immunisation of sheep with partially purified 35 kDa antigen resulted in a significant reduction of egg count following challenge with T. colubriformis, indicating the potential usefulness of this antigen in a vaccine.
A monoclonal antibody designated PAB-1 was prepared against the larval surface antigen. MAb PAB-1 and sheep mucus antibody both recognised the 35 kDa T. colubriformis larval antigen and also cross reacted with an antigen of similar molecular weight on blots of L3 extracts of the parasitic nematodes Haemonchus contortus and Ostertagia circumcincta; and with a 22 kDa antigen on blots of L3 antigens extracted from Cooperia curticei and Nematodirus spathiger. This indicated that a common surface antigen with immunising potential was present on other nematode species and could be identified by mAb PAB-1. The 35 kDa larval antigen and related molecules are likely to be novel targets for host immunity and can thus be utilised in a vaccine or other immunotherapy against nematode infections.
Monoclonal antibody PAB-1 can be used to immunopurify the surface antigen by standard affinity chromatography techniques. Monoclonal antibody PAB-1 coupled to a solid phase support such as agarose or sepharose binds the surface antigen from a crude extract of L3. The surface antigen can be eluted from the antibody matrix using a low pH buffer and shown to be substantially pure by SDS PAGE. The surface antigen purified in this manner is detected by immunoblotting against sheep antibody from immune mucus and can be stained by methods used for detecting carbohydrates.
The 35 kDa larval antigen and related molecules are known to have a predominantly carbohydrate structure. This is because the antigen is resistant to digestion by proteinase K: does not stain in gels treated with sensitive protein stains; does stain with carbohydrate stains and can be labelled with carbohydrate labelling reagents such as biotin-hydrazide.
Accordingly, mAb PAB-1 may be useful in identifying and isolating the surface antigen for development into a vaccine or other immunotherapy against nematode infections.
Serum and intestinal mucus from sheep infected with T. colubriformis contains antibody that recognises the 35 kDa larval antigen and related molecules. Accordingly, as the presence of antibody to the larval antigen indicates exposure to the parasite, monoclonal antibody PAB-1 may be useful as a diagnostic tool for the identification of infected animals.
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